Project Summary Type 2 immune responses are critical in driving lung inflammation, which involves both innate and adaptive cell types. When exposed to allergen, infectious pathogen, or other stimuli, lung epithelial cells produce cytokines such as IL-33, IL-25, and TSLP, and these alarmins promote the development of innate and adaptive responses to drive the production of type 2 cytokines including IL-4, IL-5, and IL-13, which further causes the recruitment, infiltration, proliferation and activation of other effector cells, and subsequent lung inflammation. Chronic type 2 cytokine production is also linked to the development of tissue remodeling and lung fibrosis. Macrophages, particularly tissue- resident macrophages, are critically involved in these processes to maintain tissue homeostasis. However, the detailed mechanisms of their function and regulation are far from clear. We recently initiated preliminary studies by generating conditional knockout mice by crossing VHLf/f with Lyz2cre and obtained quite unexpected results in that conditional deletion of VHL resulted in ameliorated lung inflammation, and decreased accumulation of innate lymphoid type 2 (ILC2) cells, whereas loss of Itch E3 ligase in myeloid cells results in excessive lung inflammation upon antigen/chemical exposure, accompanied by increased ILC2 cells. These preliminary studies thus allow us to postulate a central hypothesis that the E3 ligases VHL and Itch play essential roles in type 2 immune responses by regulating lung macrophages via targeting ILC2 cells. Here we would like to test this central hypothesis by proposing the following Specific Aims: 1) to study VHL in lung inflammation; and 2) to study Itch in the regulation of macrophages. The proposed studies address fundamental questions in basic immunology in general, and specific questions of the protein ubiquitination in lung inflammation and fibrosis via regulating macrophages in particular. The expected results will significantly advance our knowledge about the ubiquitination pathway in the regulation of lung inflammation, and will eventually help in the design of potential therapeutic intervention for human lung inflammatory and fibrotic diseases.